Homeostasis of the CNS is maintained within strict limits by anatomical and immunological barriers that restrict access of pathogens, solutes, and to an extent, immune cells, to the brain parenchyma. This review focuses on two of these barriers in particular; the blood–cerebrospinal fluid (CSF) barrier and blood brain barrier (BBB), which prevent the exchange of cells and solutes between the blood and CSF or brain parenchyma, respectively. The BBB is comprised of specialized endothelial cells of the cerebral microvasculature, surrounding pericytes, and astrocytic endfeet, while the blood–CSF barrier is largely made up of the fenestrated endothelium of the choroid plexus. In addition to these anatomical barriers, the expression of chemokines and chemokine receptors at the BBB and blood-CSF barrier serves as an immunological checkpoint and prevents (during non-inflammatory/homeostatic conditions) or promotes (during neuroinflammation) the infiltration of circulating leukocytes into the deeper CNS parenchyma and ventricular or subarachnoid CSF spaces (Figure 1).

As in peripheral lymphoid tissues, expression of chemokines and their cognate receptors within the CNS are highly regionalized and regulated in a tissue-dependent manner (Réaux-Le Goazigo et al., 2013). In particular, endothelial cell chemokines expressed at the BBB can translocate from the abluminal to luminal surfaces of post-capillary venules, thereby exerting effects on circulating leukocytes. Thus, a critical aspect of chemokine function at the BBB, as in peripheral tissues, is their localization along endothelial cell surfaces and binding to extracellular matrix proteins and glycosaminoglycans (GAGs). The discovery of chemokines’ ability to direct leukocyte migration was largely informed through in vitro studies of the movement of leukocytes towards increasing concentrations of solubilized chemokines (Zachariae, 1993). These analyses perhaps inappropriately fostered the notion that within tissues, leukocytes similarly respond to soluble chemokine gradients; however it is now recognized that the extracellular matrix and GAGs localize and concentrate chemokines, preventing their rapid diffusion and loss of chemotactic effects (Hamel et al., 2009). In particular, chemokines have been shown to bind with high affinity to heparan sulfate chains of heparan sulfate proteoglycans, immobilizing chemokines and leading to the formation of chemokine gradients on endothelial surfaces (Johnson et al., 2005; Parish, 2006).

The concept of immune privilege was originally conceived as a result of experiments that found antigenic material, including foreign tumors and tissue grafts, failed to elicit a systemic, T cell-mediated immune response when implanted into the CNS parenchyma (reviewed in Galea et al., 2007). While the term immune privilege implies an absence of immunological response within the CNS, it is now recognized that CNS immune privilege is not absolute but rather very elaborately controlled. Several cellular and molecular components that comprise the CNS barriers are responsible for limiting the immune response under homeostatic conditions.

Microglia and perivascular macrophages are critical components of CNS immune surveillance and protection. While microglia share morphology and many functions with perivascular macrophages, their ontogeny differs. Microglia are myeloid phagocytes (Nimmerjahn et al., 2005) derived from the yolk sac during early development (Ginhoux et al., 2010; Kierdorf et al., 2013) and are found throughout the CNS of adults. Once activated, microglia initiate a classical innate immune response, similar to that elicited by peripheral macrophages, and facilitate activation of adaptive immunity, secreting inflammatory cytokines and presenting antigen(s) to reactive lymphocytes. In adults, perivascular macrophages originate from stem cell niches in the bone marrow and localize to perivascular spaces, confined by the BBB around blood vessels, via the circulation (Neumann and Wekerle, 2013). Perivascular macrophages function similar to peripheral macrophages, and thus they are crucial for antigen presentation to and reactivation of lymphocytes, making them a critical component of CNS-defense against invading pathogens. Further, elimination of these perivascular cells enhances responses to inflammatory stimuli, including LPS, suggesting that perivascular macrophages may have a role in controlling initial host-pathogen responses within the CNS (Serrats et al., 2010). While a repertoire of chemokine and chemokine receptors are expressed by microglia and perivascular macrophages, their function with regard to antigen presentation is not well understood. However, it is likely that chemokines underlie the localization of these cells within their respective CNS compartments. Together, microglia and perivascular macrophages form another layer of CNS protection, comprising a cellular barrier to facilitate protection against invading pathogens as well as immune-mediated bystander CNS injury.

The choroid plexus and the meninges represent pivotal modified cellular barriers between the blood and CSF or parenchymal compartments, respectively. The choroid plexus is largely considered a circumventricular organ, localized in the ventricles, and constitutes one of the interfaces between the blood and the CSF (Strazielle and Ghersi-Egea, 2000; Schulz and Engelhardt, 2005). The epithelial cells of the choroid plexus secrete CSF and thus largely contribute to brain homeostasis, adjusting intracranial volume, buffering extracellular solutes, and supplying cells of the CNS with micronutrients (Strazielle and Ghersi-Egea, 2000). The vascular endothelial cells of the choroid plexus are unique from those of the BBB as they lack tight junctions, more readily enabling diapedesis of cells. Further, as opposed to postcapillary venules within the parenchyma, which require cells to traffic across two basement membranes, meningeal capillaries have only a one-layer structure (Wilson et al., 2010). In addition to representing points of access for circulating immune cells, the choroid plexus and the meninges also host a local population of antigen presenting cells. In rats and humans, choroid plexus- and meninges-associated dendritic cells (DCs) have been identified, which express major histocompatibility class II (MHC II) and present antigen to circulating lymphocytes (McMenamin, 1999). These DCs are known to sample the environment by extending processes between adjacent choroid plexus epithelial cells (Serot et al., 2000), making the choroid plexus and the meninges major sites of immunosurveillance (Figure 1).

The localization of DCs in close proximity to the vessels of the choroid plexus and meninges suggests that these cells express a set of chemokines that limit their mobility out of these compartments. Further, DC turnover dictates that these cells exhibit temporal expression of chemokine localizing cues to facilitate their egress from the circulation and into the choroid plexus and meningeal compartments (Chinnery et al., 2010). DCs are known to express several chemokine receptors including CXCR3, CCR6, CCR7, and CXCR4 as well as some others, depending on their stage of maturation (Charles et al., 2010), and numerous chemokines have been identified in recruiting DCs into the brain parenchyma during neuroinflammation as well as prion disease, viral encephalitis, brain ischemia, parasitic and bacterial CNS infections (Clarkson et al., 2012). However, it remains unclear which chemokines are involved in maintaining DCs within CNS compartments during immune surveillance. Under homeostatic conditions, the presence of DCs in the stroma of the chroroid plexus as well as meningeal blood vessels suggest a role for chemokines that are constitutively expressed at these locations, such as CCL19, CCL20 and CCL21 (Figure 2). Recent studies have also demonstrated vessel-associated, DC-like cells in the CNS perivascular space that extend cell processes into the basement membrane of the glia limitans in the absence of inflammation. These DC-like cells expressed CD11c, but not MHC II (Prodinger et al., 2011). The authors of this study speculate that these cells may represent a subpopulation of microglia, cells of the monocytic lineage, or immature or quiescent DCs capable of recognizing and presenting antigen. Since these cells extend processes into the glia limitans, it is possible that they are capable of sampling and presenting antigen within the perivascular space. It is possible that abluminally expressed CXCL12, which binds to CXCR4 expressed by DCs, at the BBB may be important in retaining these cells within close proximity to the microvasculature, allowing interactions with surveying T cells, but preventing access to deeper parenchymal tissue.

Given that mature DCs up-regulate expression of cognate chemokine receptors, including both CCR7 and CXCR4 (Sallusto et al., 1998), it is possible that expression of these receptors localize mature DCs to the chroroid plexus (via CCL21) and BBB (via CCL19 and CXCL12), thus facilitating interactions between circulating lymphocytes and these antigen presenting cells. Antigen presented by mature DCs and recognized by T cell receptors (TCRs) results in heterodimerization between TCRs and CXCR4 and is necessary to initiate activation, cytokine secretion, and T cell migration. Further, CXCL12 has been shown to enhance T cell responses via costimulation of the TCR (Smith et al., 2013), suggesting that CXCL12 expressed in the perivascular niche may play an important role in mediating TCR activation during antigen presentation. Furthermore, heterodimerization of CXCR4 and TCR (Kremer et al., 2011) triggers TCR signaling via β-arrestin-1 that results in down-regulation of CXCR4 (Schneider et al., 2009; Fernández-Arenas et al., 2014), perhaps limiting CXCL12 action at the BBB and preventing prolonged retention of T cells within the perivascular space (Figures 1 and 2).

The chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1), is expressed as three alternatively spliced isoforms (α, β, and γ). Within the CNS, the expression patterns for CXCL12 is widespread and includes the cortex, olfactory bulb, hippocampus, cerebellum, meninges, and the endothelium of the BBB (β and γ isoforms). Further, expression of CXCR4, the receptor for CXCL12, has been detected in numerous cell types in the CNS including astrocytes, microglia, oligodendrocytes, neurons, and endothelial cells of the BBB (van der Meer et al., 2000; Stumm et al., 2002). Until recently it was believed that CXCL12 mediated its effects exclusively via interactions with CXCR4, however the receptor CXCR7/ACKR3 (Atypical chemokine receptor 3), formerly the orphan receptor RDC1, has now been shown to bind CXCL12 as well as CXCL11 (Burns et al., 2006). While CXCR7/ACKR3 (CXCR7) possesses homology with conserved domains of G-protein coupled receptors and is structurally similar to other CXC receptors, ligand binding does not initiate typical intracellular signaling pathways but instead results in β-arrestin recruitment and MAP kinase activation (Rajagopal et al., 2010; Odemis et al., 2012). One function of CXCR7 appears to be its ability to act as a scavenger receptor for both CXCL12 and CXCL11, mediating uptake and degradation of these ligands, and thus regulating extracellular chemokine concentrations (Boldajipour et al., 2008; Naumann et al., 2010). The ability of CXCR7 to act as a sink for CXCL12 may have implications for controlling chemokine gradients and directing hematopoetic cells, leukocytes and other cell subsets to peripheral lymphoid tissues as well as the CNS. CXCR7 expression within the CNS of rats was detected via in situ hybridization, with CXCR7 mRNA transcripts identified in the ventricular ependyma, the choroid plexus, neuronal and astroglial cells as well as cells of the vasculature (Schönemeier et al., 2008a,b). Interestingly, studies have identified the endothelium of the BBB as a source of constitutive expression of CXCL12 and CXCR4 as well as CXCR7, suggesting a role for this chemokine/receptor axis in regulating immune cell trafficking at the BBB during homeostasis.

These initial observations of CXCL12/CXCR4 expression by BBB endothelial cells were expanded by McCandless et al. who demonstrated the importance of CXCL12 expression and polarization at the BBB in the perivascular localization of infiltrating mononuclear cells (McCandless et al., 2006). These studies determined that CXCL12 protein is normally localized along the abluminal surface of endothelium within the CNS of mice and humans. During the autoimmune diseases experimental autoimmune encephalomyelitis (EAE) in mice and multiple sclerosis (MS) in humans, CXCL12 localization shifts toward a more luminal expression pattern, which is accompanied by increased parenchymal entry of CXCR4 positive mononuclear cells. Loss of CXCL12 polarity and luminal display of this chemokine was also associated with the detection of activated CXCR4 on leukocytes within the blood, suggesting that relocation of CXCL12 in this manner not only promotes the egress of leukocytes from perivascular spaces but also increases their capture and translocation across the BBB. These results suggest that abluminal expression of CXCL12 at the CNS vasculature during homeostatic conditions is a component of immune privilege essential for limiting extravasation of circulating leukocytes across endothelial barriers, while restricting immune cells to the perivascular space, limiting their access to parenchymal tissues (Figure 2).

The intensity of immune responses in the CNS increases with proximity to the ventricles of the brain (Matyszak and Perry, 1996), and materials implanted within the subarachnoid space and meninges are likewise capable of eliciting a robust immune response. These observations suggest that the ventricular and subarachnoid CSF may function as sites of physiological immune surveillance. Consistent with this, cellular infiltrates that accumulate within the meningeal membranes during neuroinflammatory events have been observed to arrange in formations resembling secondary lymphoid structures (Howell et al., 2011), while infiltrates within the parenchyma do not exhibit the features of lymphoid neogenesis.

At one time, loss of neurons was thought to be irreversible in the adult brain; we now know that generation of replacement cells is an ongoing process in rodents and humans (Kuhn et al., 1996; Eriksson et al., 1998). Adult neurogenesis occurs in localized “neurogenic niches” from precursors that reside adjacent to the lateral ventricles, in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the hippocampus (Gage, 2000; Doetsch and Scharff, 2001; Alvarez-Buylla and Lim, 2004; Zhao et al., 2008; Sanai et al., 2011). Stem cells require extracellular signals produced by the CNS milieu to regulate their ability to self-renew, proliferate, and differentiate (Sanai et al., 2011). Cues within these local microenvironments perpetuate new neurons and facilitate their integration into the existing brain circuitry (van Praag et al., 2002; Zhao et al., 2008). Neural progenitor cells (NPCs) give rise to adult neurons, astrocytes, and oligodendrocytes, and can be classified into several lineages. GFAP⁺ astrocyte-like type B cells line the lateral ventricle and extend processes into the SVZ plexus blood vessels (Mirzadeh et al., 2008; Shen et al., 2008; Tavazoie et al., 2008). During lineage progression, type B cells give rise to transit amplifying type C cells (Pastrana et al., 2009). Type C cells rapidly divide and give rise to type A neuroblasts, which proliferate as they migrate in chains along blood vessels (Shen et al., 2008; Tavazoie et al., 2008). The endothelial cells that make up the BBB vasculature serve as neurogenic “highways”, mediating progenitor cell trafficking and differentiation by providing external signage as guidance cues.

The CNS endothelium is essential for maintenance and homeostasis of the neural progenitor pool. In the adult, bone marrow-derived endothelial progenitor cells (EPCs) express CXCR4 and respond to CXCL12 as well as other cytokines to augment neovascularization for restoration of homeostasis following CNS injury (Zhang et al., 2002; Zheng et al., 2007; Yamaguchi et al., 2003). These epithelial cells directly or indirectly give rise to all the neurons, astrocytes, and oligodendrocytes in the adult brain (Deverman and Patterson, 2009). Not only does the endothelium give rise to CNS progenitors, the neurogenic niche is localized around the vasculature. Approximately 47% of dividing progenitor (type B cells) and 46% of transit amplifying (type C) cells are located within 5 microns of the endothelium (Shen et al., 2004, 2008). These progenitors directly contact the vessels of the SVZ in areas devoid of astrocyte end-feet and pericyte coverage, suggesting the vasculature endothelium, in particular, is an essential matrix and source of external cues for NPCs (Shen et al., 2008; Teng et al., 2008).

Chemokines expressed by the vasculature in the adult CNS are dynamically or constitutively regulated to provide migratory, proliferative, or differentiation cues to neurons and glia (Deverman and Patterson, 2009). Similar to hematopoietic progenitor cells in the bone marrow, in which CXCR4-CXCL12 signaling maintains the progenitor pool (Sugiyama et al., 2006), proliferative SVZ progenitor cells home to endothelial cells in the CNS in a CXCL12- and CXCR4-dependant manner under physiologic conditions. Kokovay et al. demonstrated that in early progenitor and transit amplifying cells, epidermal growth factor receptor and α6 integrin is up-regulated downstream of CXCL12 binding, enhancing the ability of activated NPCs to bind laminin on the CNS vasculature (Figure 3). Further, they showed that CXCL12 regulates the migration of neuroblasts from the SVZ (Kokovay et al., 2010), suggesting that endothelial CXCL12 can regulate progenitor cell occupancy of and departure from the vasculature niche of the adult SVZ. Similarly, in the SGZ, Schultheiss et al. have recently demonstrated that neuronal-committed progenitor cells express CXCR4 and that CXCR4 is phosphorylated in a CXCL12-dependent fashion (Figure 3). Further, deletion of CXCR4 in NPCs of adult mice resulted in reduced neurogenesis, specifically, a reduction in Sox2⁺ early progenitors, NeuroD⁺ neuronal-committed progenitors, and doublecortin⁺ immature neurons was observed (Schultheiß et al., 2013). Together, these studies suggest that CXCL12-mediated CXCR4 activation is required for maintenance of NPCs in neurogenic zones of the adult CNS.

Multiple pathways are known to regulate CXCR4 activation in neurogenesis. During development, chemokines position neural progenitors in the SGZ such that they are exposed to a range of neurogenic factors, including Wnt and Sonic hedgehog (Shh; Klein et al., 2001; Machold et al., 2003). These factors are critical for the maintenance of adult neurogenesis as Shh promotes proliferation of NPCs (Machold et al., 2003) and manipulation of the Wnt pathway nearly abolishes neurogenesis in the adult hippocampus (Lie et al., 2005). It is now known that CXCR4 is a downstream target of Wnt signaling, suggesting that Wnt induces CXCL12-mediated processes in NPCs via receptor regulation (Choe and Pleasure, 2012).

The alternate receptor for CXCL12, CXCR7, has also been shown to have a prominent role in adult neurogenesis. Zhu et al. demonstrated that both CXCR4 and CXCR7 are required for the survival of human NPCs. While CXCR4 is broadly expressed on the surface of human NPCs, CXCR7 was primarily localized in early endosomes, quickly trafficking to the plasma membrane to mediate CXCL12 endocytosis. Treatment of human NPCs with exogenous CXCL12, however, led to CXCR4/CXCR7 colocalization and downstream ERK1/2 signaling, which was shown to be essential for NPC survival (Zhu et al., 2012). In another study, CXCR7 expression regulated the migratory behavior of early neurons in the forebrain. CXCR7, but not CXCR4, was expressed by olfactory interneuron precursors, and down-regulation of CXCR7 impacted the ability of the precursors to integrate into the rostral migratory stream, the pathway to the olfactory bulbs (Tiveron et al., 2010). These studies suggest that CXCR7 has a prominent role in adult neurogenesis both in the context of and independent of CXCR4 signaling (Figure 3).

The CXCL12/CXCR4/CXCR7 axis has multiple roles in the homeostasis of adult neurogenesis. However, it is becoming increasingly clear that this axis may be up-regulated following injury including stroke (Hill et al., 2004; Wang et al., 2012), traumatic brain injury (Israelsson et al., 2008), or demyelination (Carbajal et al., 2010, 2011; Patel et al., 2010, 2012; Williams et al., 2014) to generate replacement cells and restore normal CNS function.

Following homology cloning, phylogenetic analysis revealed that CXCL14 is one of the oldest chemokines (Huising et al., 2004), yet its functions are relatively unknown. Considered a homeostatic chemokine, CXCL14 (BRAK; breast and kidney derived) is constitutively expressed in many regions of the brain (Huising et al., 2004) including the cortex, basal ganglia, septum, hippocampus, and hypothalamus (Banisadr et al., 2011; Yamamoto et al., 2011). It is thought to have an opposing role to CXCL12 (Banisadr et al., 2011; Tanegashima et al., 2013) due to its ability to bind a shared receptor, CXCR4 (Tanegashima et al., 2013). CXCL14 is highly expressed in many regions of the adult brain, including the hippocampus, and may regulate synaptic inputs to adult NPCs (Banisadr et al., 2011). The early development of NPCs within the SGZ is regulated by excitatory GABAergic synaptic inputs that promote synaptic maturity (Ge et al., 2007; Ma et al., 2009). These newborn neurons mature and are synaptically integrated into the dentate gyrus of the hippocampus (van Praag et al., 2002). GABAergic synapses on adult NPCs are sensitive to CXCL12 and CXCL14, which enhance (Bhattacharyya et al., 2008) and inhibit the effects of GABA (Banisadr et al., 2011), respectively. These findings suggest that CXCL12 and CXCL14 work to regulate hippocampal integrity in mature mammals. This is consistent with experiments using a CXCR4 antagonist in adult mice where blockade of CXCR4 signaling impaired recognition and memory (Parachikova and Cotman, 2007). Taken together, these studies suggest that CXCL12 and CXCL14 have an opposing role, regulating NPC responses to synaptic stimulation, and maintaining balance in homeostatic NPC turnover in the adult brain.

CX₃CL1 (fractalkine) is much longer than most chemokines (373 vs. ∼80 AAs) and also exists in two forms: a 95 kDa membrane-bound form with an N-terminal chemokine domain, a glycosylated mucin-like stalk, a hydrophobic transmembrane region and an intracellular C-terminal domain; and a 70 kDa soluble form that contains only the N-terminal chemokine domain. The soluble chemokine domain of CX₃CL1, when cleaved, can act as a signaling molecule (Chapman et al., 2000), inducing chemotaxis in T cells and monocytes (Hermand et al., 2008), whereas its membrane-tethered mucin stalk can serve as a cell adhesion molecule, via binding of the CX₃CL1 receptor, CX₃CR1 (Haskell et al., 1999). Under physiologic conditions, CX₃CL1 is highly expressed by a variety of neurons throughout the CNS (Hatori et al., 2002), with especially high levels in hippocampal neurons (Sheridan and Murphy, 2013). Neurons and microglia both express its receptor, CX₃CR1 (Hatori et al., 2002), which regulates memory formation and synaptic plasticity via direct effects on glutamatergic synapses (Hoshiko et al., 2012; for an extensive review, see Sheridan and Murphy, 2013).

CX₃CL1 has been shown to play a key role in maintaining adult neurogenesis via indirect mechanisms that modify the CNS microenvironment. CX₃CL1 normally limits microglial activation and expression of proinflammatory cytokines including IL-1β, IL-6, and tumor necrosis factor (TNF)-α (Bachstetter et al., 2011; Rogers et al., 2011), which act directly on NPCs (Monje et al., 2003; Iosif et al., 2006; Koo and Duman, 2008). Thus, both genetic and antibody-based blockade of CX₃CR1 signaling attenuates the inhibition of microglial activation and impacts hippocampal neurogenesis in adult animals. With age, there is an increase in activated microglia, which can promote an inflammatory milieu (Gemma et al., 2007) and contribute to age-related declines in neurogenesis (Rao et al., 2006; Ben Abdallah et al., 2010). Both exogenous CX₃CL1 or IL-1R antagonist reverse this decline in aged animals. In addition, cleaved CX₃CL1 acts as a sensor for neuronal stress, which stimulates microglia to phagocytose excitotoxic neurons (Noda et al., 2011). Given the many facets in which CX₃CL1/CX₃CR1 signaling works to inhibit inflammation and maintain a milieu skewed towards quiescence, it is probable that CX₃CL1 contributes to the preservation of an optimal neurogenic niche for the development, proliferation, and integration of NPCs within the adult CNS.

Taken together, CX₃CL1 plays an important role in maintenance of homeostasis in the adult CNS by mediating neuron-microglia interactions during physiologic conditions. Following CX₃CR1 activation, microglia are known to remove excess neurons and support maturing synapses (Hoshiko et al., 2012; Cunningham et al., 2013; Lenz et al., 2013; Ueno et al., 2013), eliminate apoptotic neural progenitors during adult hippocampal neurogenesis (Sierra et al., 2014), and remodel neuronal circuitry during learning and memory processes (Schafer et al., 2012; Parkhurst et al., 2013). Unmanipulated adult mice deficient in CX₃CR1 had a reduction in “synaptic multiplicity”, in which fewer boutons synapsed with more than one postsynaptic spine on a single dendrite. This resulted in reduced connectivity strength between regions of the hippocampus (Zhan et al., 2014), suggesting that CX₃CL1 is required for adult hippocampal plasticity. It is clear that CX₃CL1 is crucial in maintaining homeostasis in the adult CNS; however mechanisms downstream of membrane-bound CX₃CL1- CX₃CR1 binding, as pertains to normal physiology, remain to be fully elucidated.